Not Applicable.
(1) Field of the Invention
The present invention relates to an isolated feline calicivirus and mutants thereof isolated from the urine of a cat with lower urinary tract disorder which is useful for providing a vaccine for immunizing a cat against feline calicivirus. The present invention also relates to a nucleic acid clone which encodes the capsid protein of the isolated feline calicivirus. The present invention further relates to a live or killed vaccine comprising the isolated feline calicivirus or mutants thereof, a subunit vaccine comprising the capsid antigen of the isolated feline calicivirus, a nucleic acid vaccine comprising a nucleic acid clone of the isolated feline calicivirus, and a recombinant virus vector vaccine comprising nucleic acid encoding the capsid antigen of the isolated feline calicivirus. The present invention also relates to a method for isolating feline calicivirus from urine and to assays for diagnosing cats infected with feline calicivirus.
(2) Description of Related Art
Feline calicivirus (FCV) is a small RNA virus that has been implicated in a variety of diseases in cats. The virus possesses a single-stranded RNA genome that is the plus-strand which is also polyadenylated. The molecular biology of caliciviruses is described in a review article by Clarke and Lambden in J. Gen. Virol. 78: 291-301 (1997).
FCV is generally known to cause disease characterized by conjunctivitis, rhinitis, tracheitis, pneumonia and by vesicularization/ulceration of the epithelium of the oral cavity and also by fever, anorexia, lethargy, stiff gait, and sometimes nasal and ocular discharge. Morbidity is high, mortality may reach 30%, and recovery is followed by a prolong carrier state. However, the type of disease symptoms caused by FCV depends on the strain of FCV involved, the severity of exposure, and the type of response to the FCV by the infected cat. Thus, some strains of FCV will produce little or no disease whereas other strains, such as various low virulence strains, produce no or only moderate pyrexia but may produce ulcers of the mouth and high virulence strains produce pyrexia, anorexia, depression, dypspnea, or polypnea and sometimes death, particularly, in neonatal cats. In addition to ulcers of the mouth, which may or may not occur, pneumonia may also be produced.
Disorders of the lower urinary tract of cats, characterized by hematuria, dysuria, pollakiuria, and periuria (urination in inappropriate locations), affect over half a million cats in the United States annually. As with all species, feline lower urinary tract disorders (LUTD) may result from diverse causes which may be single, multiple and interacting, or unrelated. Identified causes of feline LUTD include bacterial, fungal, and parasitic urinary tract infections; uroliths; bladder neoplasms; connective tissue strictures; lesions of the prostrate gland; and extraluminal masses of the urethra. The specific underlying cause or causes of clinical signs remain unknown in a large percentage of the naturally occurring cases of feline LUTD. In humans, hemorrhagic cystitis has been associated with a number of viral agents such as adenovirus, herpesvirus, polyomaviruses, influenza virus, and retroviruses. In cats, herpesviruses, retroviruses, and caliciviruses have at one time or another been implicated as potential causative agents in the etiopathogenesis of feline idiopathic LUTD.
Calicivirus was first implicated as a potential -causative agent in LUTDs over 30 years ago, when investigators at Cornell University isolated a calicivirus from urine obtained from a Manx cat with spontaneous urethral obstruction. Experimental induction of urethral obstruction in 80% of conventionally reared cats following urinary bladder, aerosol, or contact exposure with the Manx-isolated calicivirus directly supported the concept that calicivirus was an etiological agent for some forms of LUTD. Despite these encouraging results, the failure to re-isolate feline calicivirus from urine past the fourth day post-infection, the lack of significant serum-neutralizing antibody responses to the Manx-isolated virus, and isolation of a syncytium-forming virus from all obstructed experimental cats prompted investigators to hypothesize that feline calicivirus was not a primary causative agent of LUTD. Rather, it was hypothesized that feline calicivirus incited other latent viruses present in the urinary tract to induce urethral obstructions. The Manx-isolated calicivirus was not preserved and is no longer available, and investigators have not been successful in isolating another calicivirus from urine.
Since the discovery of FCV, FCV has been the object of numerous attempts to provide a vaccine which would protect cats against FCV infection. In U.S. Pat. Nos. 3,937,812 and 3,944,469 to Bittle et al. the development of the attenuated calicivirus F-9 vaccine strain is disclosed. The vaccine is useful for immunizing cats against respiratory infections caused by feline calicivirus. However, as shown in U.S. Pat. No. 4,522,810 to Pedersen, the F-9 vaccine strain does not protect cats against all field strains of FCV, in particular, field strain FCV-2280. Therefore, the Pedersen patent discloses FCV vaccines which consist of either live attenuated or killed FCV-2280. Pedersen does not disclose whether FCV-2280 can protect cats against a broad range of FCV field strains.
More recently, it has been observed that vaccines comprising FCV F-9 under certain field and experimental conditions can induce clinical signs of infection in vaccinated cats. To address this problem, U.S. Pat. Nos. 5,716,822 and 5,718,901 to Wardley disclose subunit vaccines consisting of the capsid polypeptide from FCV strain CFI/68 FIV. Also disclosed are transfected cells containing cDNA encoding the capsid polypeptide and recombinant baculovirus, feline herpesvirus, and vaccinia virus vectors containing the cDNA encoding the capsid polypeptide. Both the transfected cells and the baculovirus vector are used to produce the capsid polypeptide which is then used to make the subunit vaccine. The subunit vaccines avoid the problem of residual pathogenicity which may be present in live FCV vaccines. The scope of protection that was afforded by the subunit vaccine was not disclosed.
Despite the availability of vaccines for immunizing cats against FCV, none of the present vaccines have been shown to be completely protective against all strains and isolates of FCV. In some cats, some of the present vaccines may even cause disease. Furthermore, because the present vaccines consist of FCV derived from FCV isolated from cats with FCV-induced respiratory disease, the ability of the current vaccines to protect cats against FCV-induced LUTD may be ineffective. Therefore, there is a need for a vaccine that provides feline calicivirus particles or particular peptides for immunizing cats against FCV-induced LUTD.
The present invention provides isolated feline caliciviruses, FCV-U1 and FCV-U2 and mutants thereof, which were isolated from the urine of cats with lower urinary tract disorders. The present invention further provides nucleic acid clones of FCV-U1 and FCV-U2. In particular, the present invention provides a clone which encodes the capsid antigen of FCV-U1 or FCV-U2. The present invention is useful for providing a live or killed virus vaccine which comprises FCV-U1 or FCV-U2 or mutants thereof, a subunit vaccine which comprise the capsid antigen of FCV-U1 or FCV-U2 or immunogenic fragment thereof, a nucleic acid vaccine comprising a nucleic acid clone which encodes the capsid antigen of FCV-U1 or FCV-U2 or immunogenic fragment thereof, and a recombinant virus vector vaccine which comprises nucleic acid encoding the capsid antigen of FCV-U1 or FCV-U2 or immunogenic fragment thereof. The present invention also provides a method for isolating feline calicivirus from urine and an assay for diagnosing cats infected with feline calicivirus.
Thus, the present invention provides a vaccine for immunizing cats against feline calicivirus comprising FCV-U1 or FCV-U2 or mutants thereof, in an effective amount to produce an immune response, and a pharmaceutically acceptable carrier. Optionally, the vaccine can further include an adjuvant. The FCV-U1 or FCV-U2 or mutant thereof is provided as a live vaccine, preferably attenuated, or it can be provided as an inactivated vaccine. In either the live, attenuated, or inactivated embodiments of the vaccine, the FCV-U1 or FCV-U2 vaccine can further include at least one other feline calicivirus strain, preferably selected from the group consisting of FCV-F9, FCV-LLK, FCV-M8, FCV-255, and FCV-2280, in addition to the FCV-U1 or FCV-U2 or mutant thereof.
The present invention also provides a vaccine for immunizing cats against feline calicivirus comprising a recombinant virus vector comprising a nucleotide sequence encoding the capsid antigen or immunogenic fragment thereof selected from the group consisting of FCV-U1, FCV-U2 or both, wherein the nucleic acid sequence is operably linked to a heterologous promoter sequence, in an effective amount to produce an immune response, and a pharmaceutically acceptable carrier. In particular, a vaccine as provided wherein the recombinant virus vector is selected from the group consisting of feline herpesvirus, raccoon poxvirus, canary poxvirus, adenovirus, Simliki forest virus, sindbis virus, and vaccinia virus. In a preferred embodiment, the nucleotide sequence encoding the capsid antigen or immunogenic fragment thereof comprises the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6 or the nucleotide sequence encoding the capsid antigen or immunogenic fragment thereof comprises the nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.
Further still, the present invention provides a vaccine to immunize cats against feline calicivirus which comprises a nucleotide sequence or portion thereof encoding the capsid antigen or immunogenic fragment thereof of the calicivirus selected from the group consisting of FCV-U1, FCV-U2, or both, wherein the nucleic acid sequence is operably linked to a heterologous promoter sequence, in an effective amount to produce an immune response, and a pharmaceutically acceptable carrier. Preferably, the nucleotide sequence encoding the capsid antigen or immunogenic fragment thereof is in a plasmid, and in particular, the capsid antigen or immunogenic fragment thereof encoded by the nucleotide sequence comprises the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 or the capsid antigen is encoded by the nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.
Further still, the present invention provides a vaccine to immunize cats against feline calicivirus comprising an isolated capsid antigen or portion thereof selected from the group consisting of FCV-U1, FCV-U2, and both, in an effective amount to produce an immune response, and a pharmaceutically acceptable carrier. Preferably, the capsid antigen comprises the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6.
The present invention also provides a method for immunizing a cat against feline calicivirus comprising administering to the cat an effective dose of a vaccine selected from the group consisting of a live FCV-U1 or mutant thereof, a killed FCV-U1 or mutant thereof, a recombinant virus vector comprising a nucleotide sequence encoding a capsid antigen or immunogenic fragment thereof of FCV-U1 or mutant thereof, a nucleotide molecule comprising a nucleotide sequence encoding the capsid antigen or immunogenic fragment thereof of FCV-U1 or mutant thereof, and an isolated capsid antigen or immunogenic fragment thereof of FCV-U1 or mutant thereof, in a pharmaceutically acceptable carrier. Preferably, the capsid antigen or immunogenic fragment thereof comprises the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6 or the capsid antigen or immunogenic fragment thereof is encoded by the nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.
Further still, the present invention provides a method for immunizing a cat against feline calicivirus comprising administering to the cat an effective dose of a vaccine selected from the group consisting of a live FCV-U2 or mutant thereof, a killed FCV-U2 or mutant thereof, a recombinant virus vector comprising a nucleotide sequence encoding a capsid antigen or immunogenic fragment thereof of FCV-U2 or mutant thereof, a nucleotide molecule comprising a nucleotide sequence encoding the capsid antigen or immunogenic fragment thereof of FCV-U2 or mutant thereof, and an isolated capsid antigen or immunogenic fragment thereof of FCV-U2 or mutant thereof, in a pharmaceutically acceptable carrier.
The present invention also provides an isolated and purified culture of feline calicivirus FCV-U1 or mutant thereof deposited as ATCC PTA-3444 or an isolated and purified culture of feline calicivirus FCV-U2 or mutant thereof deposited as ATCC PTA-3445.
The present invention also provides a pure and isolated capsid antigen or immunogenic fragment thereof of feline calicivirus comprising the amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, and SEQ ID NO:6. In particular, the pure and isolated capsid antigen or immunogenic fragment thereof, wherein the amino acid sequence is encoded by the nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.
Finally, the present invention provides a nucleotide molecule encoding a feline calicivirus capsid antigen or immunogenic fragment thereof comprising the nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, and SEQ ID NO:5.
Therefore, an object of the present invention is to provide a vaccine that immunizes cats against disease caused by feline calicivirus.
These and other objects of the present invention will become increasingly apparent with reference to the following drawings and preferred embodiments.